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Cited by 30Cellular transport and secretionNeuroscience and Neuropharmacology ResearchProtein Kinase Regulation and GTPase Signaling

Post-Translational Modifications and Lipid Binding Profile of Insect Cell-Expressed Full-Length Mammalian Synaptotagmin 1

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Vrljic, Marija, Strop, Pavel, Hill, Ryan C., Hansen, Kirk C., Chu, Steven, Brunger, Axel T.·2011·Biochemistry
DOI 10.1021/bi200998yPMC PMC3217305
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Abstract

Synaptotagmin 1 (Syt1) is a Ca(2+) sensor for SNARE-mediated, Ca(2+)-triggered synaptic vesicle fusion in neurons. It is composed of luminal, transmembrane, linker, and two Ca(2+)-binding (C2) domains. Here we describe expression and purification of full-length mammalian Syt1 in insect cells along with an extensive biochemical characterization of the purified protein. The expressed and purified protein is properly folded and has increased α-helical content compared to the C2AB fragment alone. Post-translational modifications of Syt1 were analyzed by mass spectrometry, revealing the same modifications of Syt1 that were previously described for Syt1 purified from brain extract or mammalian cell lines, along with a novel modification of Syt1, tyrosine nitration. A lipid binding screen with both full-length Syt1 and the C2AB fragments of Syt1 and Syt3 isoforms revealed new Syt1-lipid interactions. These results suggest a conserved lipid binding mechanism in which Ca(2+)-independent interactions are mediated via a lysine rich region of the C2B domain while Ca(2+)-dependent interactions are mediated via the Ca(2+)-binding loops.

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  • Syt1 binds in a Ca2+-dependent manner to anionic membranes, with anionic phospholipids and synaptotagmin C2 domains together coordinating calcium.

    “Syt1 binds in a Ca2+-dependent manner to anionic membranes; during binding, anionic phospholipids and synaptotagmin C2 domains together coordinate calcium14–17.”

    From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [17]

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Authors
Vrljic, M., Strop, P., Hill, R. C., Hansen, K. C., Chu, S., & Brunger, A. T.
Affiliations
  • Howard Hughes Medical Institute (US)
  • University of Colorado Health (US)
  • Lawrence Berkeley National Laboratory (US)
  • Department of Molecular and Cellular Physiology, Department of Neurology and Neurological Science, Department of Structural Biology, and Department of Photon Science, Stanford University, Stanford, California 94305-5432, United States
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2011-10-26
Volume
50
Issue
46
Pages
9998-10012
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/bi200998y
PMC
PMC3217305
OpenAlex
W2052023774
Cited by
30
References
0
Citations by year
2023: 2 · 2022: 5 · 2019: 1 · 2018: 4 · 2017: 4 · 2016: 1 · 2015: 4 · 2014: 3 · 2013: 2 · 2012: 4
FWCI
1.38
Type
journal article
Language
en
Open access
Licence
Locations
  • Biochemistry (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Institutes of Health
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Keywords
synaptotagmin-1, post-translational modification, lipid binding, C2 domain, tyrosine nitration, Ca2+ sensor, synaptic vesicle fusion, SNARE proteins, C2B domain, insect cell expression, protein purification, protein folding
MeSH terms
Amino Acid Sequence, Animals, Cell Line, Cloning, Molecular, Glycosylation, Insecta, Models, Molecular, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Gene Expression, Sequence Alignment, Protein Structure, Secondary, Protein Folding, Lipid Metabolism, Synaptotagmin I, Rats

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